Methods for preparing a sample for nucleic acid amplification using tagmentation

ABSTRACT

Presented are methods and compositions for preparing samples for amplification and sequencing. Particular embodiments relate to methods of preparing nucleic acid-continuing cellular samples for library amplification, wherein the methods include lysing cells of the sample to form a lysate, amplifying the nucleic acids from the lysed samples, exposing the amplified nucleic acids to a solid surface, and clonally amplifying the amplified nucleic acids to generate clusters.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/163,005, filed on Oct. 17, 2018, and issued as U.S. Pat. No.10,774,367 on Sep. 15, 2020 which is a continuation of U.S. patentapplication Ser. No. 14/733,452, filed on Jun. 8, 2015, which claims thebenefit of priority to U.S. Provisional Application No. 62/171,814,filed Jun. 5, 2015 and U.S. Provisional Application No. 62/171,908,filed Jun. 5, 2015, and claims the benefit of priority to EuropeanPatent Application No. 1410196.8, filed on Jun. 9, 2014, and EuropeanPatent Application No. 1412207.1, filed on Jul. 9, 2014. The disclosuresof the above-referenced applications are hereby expressly incorporatedby reference in their entireties.

FIELD

The present invention relates to methods for preparing samples forsubsequent nucleic acid (e.g. DNA) amplification, which methods aresimpler to perform than existing methods. In particular, the presentinvention relates to methods wherein purifying nucleic acid (e.g. DNA)from a sample is not required prior to amplification.

BACKGROUND

Traditional DNA amplification methods typically require purified DNA tobe obtained prior to the amplification steps. The purification processtypically requires enzymatic digestion or lysis of cells in a cellularsample, followed by one or more separation steps to separate out the DNAfrom the cellular debris, which may include one or more washing stepsand final elution of the purified DNA into a tube ready for use in anamplification process (such as PCR). The process often takes upwards of30 minutes, typically 40 minutes or more.

Recently, Sigma has developed a so-called ‘Extract-N-Amp™ Blood PCRKit’, which contains reagents necessary to extract host genomic DNA fromwhole blood and amplify targets of interest by PCR. This extractionsystem reduces the need for purification, organic extraction,centrifugation, heating, filtration or alcohol precipitation. The kitalso includes a PCR Ready mix, especially formulated for amplificationdirectly from the extract. This formulation uses an antibody based HotStart, for specific amplification. Genomic DNA is extracted from 10 μlof whole blood by simply adding the Extraction Solution (which appearsto be potassium hydroxide) and incubating for 5 minutes at roomtemperature. The Neutralization Solution is added to the extract tocounteract inhibitory substances prior to PCR. A portion of the DNAextract is then added to the specially formulated PCR mix.

It is an object of the present invention to provide sample preparationmethods that do not require purification of DNA prior to amplification.Preferably, those methods require only simple reagents, which reducesthe time and cost burden on persons performing the preparations.

SUMMARY

In one embodiment of the present invention, there is provided a methodof preparing a sample for library amplification and subsequentamplification comprising the following steps:

(a) providing a nucleic acid-containing cellular sample;

(b) lysing cells of the sample to liberate nucleic acid from within thecells of the cellular sample, thereby forming a lysate; and

(c) amplifying the nucleic acid from the lysed samples;

wherein there is no purification of the nucleic acid from the lysedsample prior to beginning the amplification step (c).

Preferably the nucleic acid is DNA.

Preferably the sample is a clinical or non-clinical sample.

Preferably the sample is a blood sample.

Preferably the blood sample is a whole blood sample.

In one embodiment, the sample is taken from a culture. In anotherembodiment, the sample is taken from a microbiological culture (e.g., ablood culture).

Preferably the sample is a non-blood sample, such as a tissue sample(e.g. tumor, biopsy), an aspirate, etc.

Preferably the lysis reagent is water, preferably purified/distilledwater.

Preferably the lysis reagent not water. Examples may include detergents,acids, bases, enzymes.

Preferably the sample and lysis reagent are mixed together to achievemore even distribution.

Optionally an enzyme is further added to the lysate in order to disruptthe DNA structure. Preferably the enzyme is proteinase K.

Optionally, there is a neutralizing step after lysis of the cells withthe lysis reagent to inactivate the lysis reagent if required.Preferably this neutralizing step is prior to the amplification step(c). In some aspects, the neutralizing step can be considered as part ofan incubation period. The same or an additional neutralizing step canalso be performed in order to neutralize any other agent in the lysatethat may interfere with subsequent amplification steps, such asproteinase K when tagmentation is to be performed as part of theamplification process.

Optionally there is an incubation period after combining the sample andthe lysis reagent. The incubation period should be sufficient to allowlysis of a portion, preferably the majority or substantially all or all,of the cells in the sample, including their cell membranes (andpreferably including nuclear membranes), such that nucleic acid (e.g.DNA) of the cell becomes accessible for suitable amplification. Whilstincubation can occur at temperatures higher than room temperature,incubation does not necessarily imply that a raised temperature is used.Incubation can occur at or around room temperature, or at less than roomtemperature. Times for incubation can range from a couple of seconds,e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 seconds, to anumber of minutes, e.g. about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 minutes. Longer incubation periods maybe required depending on the sample and/or lysis reagent, such as about20, 30, 40, 50, 60, 70, 80, 90 minutes. Ranges of incubation times arealso permitted, involving a combination of any of the above-mentionedtimes as being lower and upper limits, respectively (e.g. about 0.5-10minutes, about 1-5 minutes, about 2-5 minutes, about 1-8 minutes, etc.).

In one aspect of the present invention, the steps of lysing the sampleand amplifying the nucleic acid contained therein is conducted in asingle pot reaction.

The lysate formed from lysing the cells can comprise all of the contentsand fragments of cell membranes etc. produced when the cell is lysed,such as e.g. the cytoplasm and its components. In the context of thepresent invention, the lysate may also be considered as being thecontents of the lysed cell excluding such things as cell membranefragments and larger cellular debris (such as organelles etc. (that e.g.have escaped lysis during the lysis step)). In other words, the lysatemay comprise the cytosol of the cell, along with lipids, proteins, andnucleic acids.

By the term “there is no purification of the nucleic acid from the lysedsample prior to beginning the amplification step (c)” it is meant thatthe nucleic acid (e.g. DNA) is not isolated or separated away from thelysate prior to initiating the amplification process (the amplificationprocess itself may of course comprise steps of purifying the nucleicacid as part of the amplification process). However, it is not meant tolimit additional steps being performed to alter or modify the nucleicacid (e.g. DNA) or its tertiary structure after lysis and beforeamplification in order that the amplification process can be carried outsuccessfully.

In one aspect of the invention, quantification of the amount of DNA inthe lysate is conducted prior to the amplification step.

In further embodiments of the present invention, the amplified DNA issequenced to ascertain its sequence. This can be done by any methodknown in the art. Preferably, it is sequenced by high throughputsequencing, such as a sequence by synthesis protocol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow diagram of an example of method of preparing awhole blood sample for targeted DNA amplification;

FIG. 2 shows a plot of clusters that pass filter for amplicons generatedby targeted DNA amplification of a whole blood sample prepared accordingto the method of FIG. 1;

FIGS. 3A and 3B show a plot of amplicon size and a plot of percent GCcontent, respectively, from a targeted DNA amplification assay performedon a whole blood sample prepared by dilution with water;

FIG. 4 shows a data table of the sequencing metrics for the targeted DNAamplification assay of FIGS. 3A and 3B;

FIG. 5 shows panels of clusters generated from amplicons generateddirectly from a whole blood sample by targeted DNA amplification;

FIG. 6 illustrates a flow diagram of an example of a method of preparinga whole blood sample for construction of a tagmented DNA library(Nextera);

FIGS. 7A and 7B show a bar graph of sequencing depth per chromosome anda plot of the size distribution, respectively, of a tagmented DNAlibrary prepared according to the method of FIG. 6;

FIG. 8 illustrates a flow diagram of an example of a method of preparingan FFPE sample for targeted amplification and subsequent sequencing;

FIG. 9A shows a bar graph of the Q score metric and a data table ofsequencing metrics for amplicons generated from DNA prepared from acolon tumor FFPE sample according to the method of FIG. 8;

FIG. 9B shows a bar graph of the Q score metric and a data table ofsequencing metrics for amplicons generated from DNA prepared from acolon tumor FFPE sample using a QiaAmp DNA purification kit;

FIGS. 10A and 10B show a data table of sequencing metrics and a plot ofamplicon clusters for the QiaAmp prepared DNA and the QuickExtractprepared DNA of FIGS. 9A and 9B; and

FIGS. 11A and 11B show a plot of amplicon size and a plot of percent GCcontent, respectively, from the direct targeted amplification of an FFPEslice (no incubation); and

FIG. 12 illustrates an example of a method of sequencing from driedblood spots;

FIGS. 13A (a data table of sequencing metrics) and 13B (a plot ofamplicon size and a plot of percent GC content) show the results of thesequencing of FIG. 12; and

FIG. 14 illustrates an example of a method of tagmentation andsequencing from blood spots with the upper panel including a wash stepin water and the lower panel omitting the wash step in water; and

FIG. 15 (a data table of sequencing metrics) shows the results of thesequencing of FIG. 14.

FIG. 16 shows a data table of sequencing metrics (top) and a graphshowing sequencing depth per chromosome (bottom) for a samplepreparation method of preparing a whole blood sample for construction ofa tagmented DNA library (Nextera) performed according to the workflowillustrated in FIG. 6.

FIG. 17 shows a data table of sequencing metrics (top) and a graphshowing sequencing depth per chromosome (bottom) for a samplepreparation method of preparing a whole blood sample for construction ofa tagmented DNA library (Nextera) performed according to the workflowillustrated in FIG. 6, but using bead-based tagmentation for thetagmentation step.

FIG. 18 shows a data table of sequencing metrics (top) and a graphshowing sequencing depth per chromosome (bottom) for a samplepreparation method of preparing a tagmented DNA library (Nextera)performed by rinsing a dried blood spot in water, followed bytagmentation (Nextera).

FIG. 19 shows a data table of sequencing metrics for three BBN samples(BBN1, 2, 3) as compared to sequencing metrics of purified gDNAcontrols.

FIG. 20 is a graph showing coverage for gene panels of interest fromCommon Genetic Disorders and UK Genetic testing network (UKGTN) forblood samples sequenced using BBN, dried blood spots, and control gDNA.Shown are median values (top two panels) and standard deviation (lowerpanel).

FIG. 21 is a graph comparing precision and recall for SNP concordancefor blood samples sequenced using BBN, dried blood spots, and controlgDNA.

FIG. 22 shows sample preparation workflows for preparing a DNA libraryfrom a whole blood sample using standard TruSeq Nano method ormodifications thereof.

FIG. 23 is a graph showing GC bias profile of libraries preparedaccording to the workflows illustrated in FIG. 22.

FIG. 24 shows graphs comparing library diversity (left panel) andsequencing efficiency (right panel) of libraries prepared according tothe workflows illustrated in FIG. 22.

FIG. 25 shows sample preparation workflows for preparing a DNA libraryfrom an FFPE sample using modifications of the workflow set forth inFIG. 8.

FIG. 26 is a data table comparing coverage uniformity obtained forsequencing libraries obtained using 4 different workflows from 3different FFPE samples.

FIG. 27 shows plots of clusters that pass filter for amplicons generatedby targeted DNA amplification of FFPE samples prepared according to twomethods of FIG. 25.

DETAILED DESCRIPTION

The invention provides methods of preparing samples for nucleic acidamplification. The nucleic acid may be DNA, or RNA. In one embodiment,the invention provides methods of preparing a blood sample for nucleicacid amplification, preferably wherein the blood sample is a whole bloodsample.

In another embodiment, the invention provides methods of preparingnon-blood samples, such as tissue samples (e.g. formalin fixedparaffin-embedded (FFPE) samples) for DNA amplification. Such tissuesamples may be tumor samples. Other samples may be biopsies, oraspirates, etc.

DNA amplification may be performed according to the methods described inthe WO2010/038042 publication, the WO2011/025477 publication, PCTapplication PCT/US2014/071263, filed Dec. 18, 2014, and/or PCTapplication PCT/EP2014/079145, filed Dec. 23, 2014, each of which isincorporated herein by reference in its entirety. Targeted DNAamplification may be used to enrich target sequences for subsequentcluster generation and sequencing.

The methods of the invention preferably use (whole) blood or non-blood(e.g. FFPE) tissue samples as sample input. The methods of the inventionobviate the need for nucleic acid (e.g. DNA) purification prior toamplification.

The invention also provides a method for tagmentation (e.g. using theNextera™ process (Illumina, Inc.)) of nucleic acid (e.g. DNA) in asample, such as a whole blood sample.

In simple terms, the present invention provides the steps of: (a)providing a nucleic acid-containing cellular sample;

-   -   (b) lysing cells of the sample to liberate nucleic acid from        within the cells of the cellular sample, thereby forming a        lysate; and    -   (c) amplifying the nucleic acid from the lysed samples;    -   wherein there is no purification of the nucleic acid from the        lysed sample prior to beginning the amplification step (c).

FIG. 1 illustrates a flow diagram of an example of method 100 ofpreparing a whole blood sample for targeted DNA amplification. Forexample, targeted DNA amplification may be performed according to themethods described in the WO2010/038042 publication, the WO2011/025477publication, PCT application PCT/US2014/071263, filed Dec. 18, 2014,and/or PCT application PCT/EP2014/079145, filed Dec. 23, 2014, each ofwhich is incorporated herein by reference in its entirety. Method 100includes, but is not limited to, the following steps.

At a step 110, a nucleic acid-containing sample is obtained or provided.This can be a blood sample, or a non-blood sample such as a tissuesample, biopsy, aspirate, etc. An example of a tissue sample might bee.g. a tumor sample. If the sample is a blood sample, then preferably itis a whole blood sample.

The quantity of sample provided will depend on the sample and on thesubsequent procedure that will be conducted on the sample. Typically,sample amounts for liquid samples may be in the region of approximately1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 μL. Such amountsare suitable for blood samples which will undergo a PCR amplificationprocess. In some aspects, the blood sample amount will be approximately10 μL. In other aspects, the blood sample amount will be approximately 2μL.

In embodiments of the present invention where a solid sample isprovided, a sufficient amount of sample should be used which willliberate sufficient nucleic acid. The skilled person will be aware ofhow to prepare a suitable amount of sample.

At a step 115, the cells in the sample (e.g. whole blood, tissue) arelysed. For example, an aliquot (e.g. 10 μL) of the whole blood sample ismixed with an amount of a lysis reagent. The lysis reagent may be anysuitable reagent for disrupting and/or solubilizing the cell membrane.

A lysis solution is one that is capable of lysing cells (e.g., bysolubilizing eukaryotic cell membranes). Preferably, the lysis solutionis one that leaves nucleic acid intact (i.e. that does not denature anucleic acid chain to an extent that the chain is disrupted toindividual nucleic acids). In one embodiment, the lysis solution cancomprise one or more detergents, one or more enzymes, or a combinationof one or more detergents and one or more enzymes, and can furtherinclude additional agents. In one embodiment, the detergent can be anon-denaturing lytic detergent, such as Triton® X-100 Triton® X-100-R,Triton® X-114, NP-40, Genapol® C-100, Genapol® X-100, Igepal® CA 630,Arlasolve™200, Brij® 96/97, CHAPS, octyl β-D-glucopyranoside, saponin,and nonaethylene glycol monododecyl ether (C12E9, polidocenol).Optionally, solubilizers can also be included, such as Brij® 98, Brij®58, Brij® 35, Tween® 80, Tween® 20, Pluronic® L64, Pluronic® P84,non-detergent sulfobetaines (NDSB 201), amphipols (PMAL-C8), andmethyl-β-cyclodextrin. Typically, non-denaturing detergents andsolubilizers are used at concentrations above their critical micelleconcentration (CMC), while denaturing detergents may be added atconcentrations below their CMC. For example, non-denaturing lyticdetergents can be used at a concentration of about 0.010% to about 10%,e.g., about 0.015% to about 1.0%, e.g., about 0.05% to about 0.5%, e.g.,about 0.10% to about 0.30% (final concentration after dilution with thesample). In another embodiment, polyoxyethylene detergent detergents maybe preferred. The polyoxyethylene detergent can comprise the structureC₁₂₋₁₈/E₉₋₁₀, wherein C12-18 denotes a carbon chain length of from 12 to18 carbon atoms and E9-10 denotes from 9 to 10 oxyethylene hydrophilichead groups. For example, the polyoxyethylene detergent can be selectedfrom the group consisting of Brij® 97, Brij® 96V, Genapol® C-100,Genapol® X-100, nonaethylene glycol monododecyl ether (polidocanol), ora combination thereof.

Enzymes that can be used in lysis solutions include, without limitation,enzymes that are considered membrane-fouling materials (e.g., proteinaseXXIII, neuraminidase, polysaccharidase, Glucanex®, and Pectinex®). Otheradditives that can be used include, without limitation, reducing agentssuch as 2-mercaptoethanol (2-Me) or dithiothreitol (DTT) and stabilizingagents such as magnesium, pyruvate, and humectants.

The lysis solution can be buffered at any pH that is suitable to lysethe desired cells, and will depend on multiple factors, includingwithout limitation, the type of sample, the cells to be lysed, and thedetergent used. In some embodiments, the pH can be in a range from about2 to about 13, e.g., about 6 to about 13, e.g., about 8 to about 13,e.g., about 10 to about 13. Suitable pH buffers include any buffercapable of maintaining a pH in the desired range, e.g., about 0.05 M toabout 1.0 M CAPS.

In one example, the lysis reagent is the lysis reagent from an“Extract-N-Amp” Blood PCR kit (available from Sigma).

A suitable volume of a lysis reagent is e.g. 10 μL to 200 μL, dependingon the reagent. Volumes might be selected from approximately 10, 20, 30,40, 50, 60, 70, 80, 90, 100 μL, and will depend on the amount of sampleto be lysed.

In one embodiment, a lysis reagent (e.g. lysis reagent from an“Extract-N-Amp” Blood PCR kit (available from Sigma), e.g. potassiumhydroxide, might be used at approximately 20 μL, when 10 μL of e.g.blood is used.

Preferably the lysis reagent is water, preferably distilled water. Inone embodiment, water is used preferably in an amount of 90 μL, where a10 μL sample e.g. blood is provided. The skilled person will be able tovary the amount of water used dependent on the sample size in accordancewith their general knowledge and usual lab practices. For example, avolume of 12 μL water may be mixed with a 2 μL aliquot of whole blood.

After the lysis reagent (e.g. water) has been added to the cell sample(e.g. blood sample), the mixture may optionally be mixed (e.g. via avortex mixer, or by shaking by hand). Mixing allows the lysis reagentand sample to be evenly distributed, so that the sample is lysed asequally as possible. Mixing may occur for a few to a number of seconds(e.g. 5 s to 60 s).

It should be appreciated that the lysis reagent may be added to thesample, or the sample may be added to the lysis reagent.

After the lysis reagent and sample have been combined and optionallymixed, there is an incubation period. This allows the lysis reagentsufficient time to lyse the cells in the sample. The optional mixingstep may also form part of the time of the incubation period.

In one embodiment, the sample and the lysis solution are mixed and thenincubated for a sufficient time for lysis and solubilization of cellmembranes to occur, e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40,50, or 60 seconds, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20minutes or longer, e.g., about 1 second to about 20 minutes, about 1second to about 5 minutes, or about 1 second to about 2 minutes. Longerincubation times may also be necessary depending on the sample and/orlysis reagent. For example, approximately 30, 40, 50, 60, 70, 80, 90minutes. The incubation time will depend on the strength of the lysissolution, e.g., the concentration of the detergent and/or enzymes. Thelysis can take place at a temperature of about 2° C. to about 45° C.,e.g., about 15° C. to about 40° C., e.g., about 30° C. to about 40° C.,room temperature, etc. In one embodiment, the lysis solution can beloaded into a syringe and the sample can then be aspirated into thesyringe such that mixing and incubation occurs within the syringe. Inone embodiment, the lysis solution can be loaded into a syringe and thesample can then be aspirated into the syringe such that mixing andincubation occurs within the syringe.

In an embodiment of the present invention, particularly where the lysisreagent is not water, the incubation time is approximately 5 minutes atroom temperature.

In a particularly preferred embodiment of the invention where the lysisreagent is water (i.e. without any other lysis reagent (e.g.detergent)), the incubation time (room temp) is approximately 2 minutes.This represents a significant time saving over using non-water detergentlysis reagents.

At a step 120, there is an optional neutralization step. This may beneeded if the lysis reagent is required to be neutralized prior to theamplification step, due to interference of the lysis reagent with theamplification process that would otherwise occur.

In preferred embodiments of the present invention, the lysis reagent isselected such that no neutralizing step is required. The use of water asa lysis reagent does not require a subsequent neutralizing step prior toamplification.

Where a neutralizing step is required, the skilled person will be wellaware of the amount of neutralizing agent required to neutralize thelysis reagent. For example, the lysis reaction can be neutralized by theaddition of a neutralization reagent from the “Extract-N-Amp” Blood PCRkit. A suitable amount of such a reagent may be approximately 180 μL.

At a step 125, an aliquot of the lysed (and optionally neutralized)blood sample is amplified by targeted DNA amplification. Any suitableamplification method may be used, and will typically employ PCR. Thepresent invention is not necessarily limited by a particularamplification process. Depending on the type of amplification methodemployed, the amount of lysed sample required for the amplificationprocess will vary accordingly. For example, an amount of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μL may besuitable for the amplification process. For example, 2, or 4 μL.

Nucleic Acid Amplification and Clustering

In some embodiments, the immobilized DNA fragments are amplified usingcluster amplification methodologies as exemplified by the disclosures ofU.S. Pat. Nos. 7,985,565 and 7,115,400, the contents of each of which isincorporated herein by reference in its entirety. The incorporatedmaterials of U.S. Pat. Nos. 7,985,565 and 7,115,400 describe methods ofsolid-phase nucleic acid amplification which allow amplificationproducts to be immobilized on a solid support in order to form arrayscomprised of clusters or “colonies” of immobilized nucleic acidmolecules. Each cluster or colony on such an array is formed from aplurality of identical immobilized polynucleotide strands and aplurality of identical immobilized complementary polynucleotide strands.The arrays so-formed are generally referred to herein as “clusteredarrays”. The products of solid-phase amplification reactions such asthose described in U.S. Pat. Nos. 7,985,565 and 7,115,400 are so-called“bridged” structures formed by annealing of pairs of immobilizedpolynucleotide strands and immobilized complementary strands, bothstrands being immobilized on the solid support at the 5′ end, preferablyvia a covalent attachment. Cluster amplification methodologies areexamples of methods wherein an immobilized nucleic acid template is usedto produce immobilized amplicons. Other suitable methodologies can alsobe used to produce immobilized amplicons from immobilized DNA fragmentsproduced according to the methods provided herein.

For example one or more clusters or colonies can be formed viasolid-phase PCR whether one or both primers of each pair ofamplification primers are immobilized.

In other embodiments, the immobilized DNA fragments are amplified insolution. For example, in some embodiments, the immobilized DNAfragments are cleaved or otherwise liberated from the solid support andamplification primers are then hybridized in solution to the liberatedmolecules. In other embodiments, amplification primers are hybridized tothe immobilized DNA fragments for one or more initial amplificationsteps, followed by subsequent amplification steps in solution. Thus, insome embodiments an immobilized nucleic acid template can be used toproduce solution-phase amplicons.

It will be appreciated that any of the amplification methodologiesdescribed herein or generally known in the art can be utilized withuniversal or target-specific primers to amplify immobilized DNAfragments. Suitable methods for amplification include, but are notlimited to, the polymerase chain reaction (PCR), strand displacementamplification (SDA), transcription mediated amplification (TMA) andnucleic acid sequence based amplification (NASBA), as described in U.S.Pat. No. 8,003,354, which is incorporated herein by reference in itsentirety. The above amplification methods can be employed to amplify oneor more nucleic acids of interest. For example, PCR, including multiplexPCR, SDA, TMA, NASBA and the like can be utilized to amplify immobilizedDNA fragments. In some embodiments, primers directed specifically to thenucleic acid of interest are included in the amplification reaction.

Other suitable methods for amplification of nucleic acids can includeoligonucleotide extension and ligation, rolling circle amplification(RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), which isincorporated herein by reference) and oligonucleotide ligation assay(OLA) (See generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and5,573,907; EP 0 320 308 B 1; EP 0 336 731 B 1; EP 0 439 182 B 1; WO90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated byreference) technologies. It will be appreciated that these amplificationmethodologies can be designed to amplify immobilized DNA fragments. Forexample, in some embodiments, the amplification method can includeligation probe amplification or oligonucleotide ligation assay (OLA)reactions that contain primers directed specifically to the nucleic acidof interest. In some embodiments, the amplification method can include aprimer extension-ligation reaction that contains primers directedspecifically to the nucleic acid of interest. As a non-limiting exampleof primer extension and ligation primers that can be specificallydesigned to amplify a nucleic acid of interest, the amplification caninclude primers used for the GoldenGate assay (Illumina, Inc., SanDiego, Calif.) as exemplified by U.S. Pat. Nos. 7,582,420 and 7,611,869,each of which is incorporated herein by reference in its entirety.

Exemplary isothermal amplification methods that can be used in a methodof the present disclosure include, but are not limited to, MultipleDisplacement Amplification (MDA) as exemplified by, for example Dean etal., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or isothermal stranddisplacement nucleic acid amplification exemplified by, for example U.S.Pat. No. 6,214,587, each of which is incorporated herein by reference inits entirety. Other non-PCR-based methods that can be used in thepresent disclosure include, for example, strand displacementamplification (SDA) which is described in, for example Walker et al.,Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S.Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res.20:1691-96 (1992) or hyperbranched strand displacement amplificationwhich is described in, for example Lage et al., Genome Research13:294-307 (2003), each of which is incorporated herein by reference inits entirety. Isothermal amplification methods can be used with thestrand-displacing Phi 29 polymerase or Bst DNA polymerase largefragment, 5′→3′ exo- for random primer amplification of genomic DNA. Theuse of these polymerases takes advantage of their high processivity andstrand displacing activity. High processivity allows the polymerases toproduce fragments that are 10-20 kb in length. As set forth above,smaller fragments can be produced under isothermal conditions usingpolymerases having low processivity and strand-displacing activity suchas Klenow polymerase. Additional description of amplification reactions,conditions and components are set forth in detail in the disclosure ofU.S. Pat. No. 7,670,810, which is incorporated herein by reference inits entirety.

Another nucleic acid amplification method that is useful in the presentdisclosure is Tagged PCR which uses a population of two-domain primershaving a constant 5′ region followed by a random 3′ region as described,for example, in Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993),incorporated herein by reference in its entirety. The first rounds ofamplification are carried out to allow a multitude of initiations onheat denatured DNA based on individual hybridization from therandomly-synthesized 3′ region. Due to the nature of the 3′ region, thesites of initiation are contemplated to be random throughout the genome.Thereafter, the unbound primers can be removed and further replicationcan take place using primers complementary to the constant 5′ region.

As can be seen in the present invention, the time savings using thesimplified method of nucleic acid preparation (e.g. approximately 2-5minutes) where no DNA purification is conducted over traditional DNApurification techniques (e.g. over 20 minutes, typically 30-120 minutes)is significant.

FIG. 2 shows a plot 200 of clusters that pass filter for ampliconsgenerated by targeted DNA amplification of a whole blood sample preparedaccording to method 100 of FIG. 1. In particular, 10 μL whole blood, 20μL lysis reagent (“Extract-N-Amp” Blood PCR kit, Sigma), 5 minuteincubation at room temp, 180 μL neutralizing reagent (“Extract-N-Amp”Blood PCR kit, Sigma), 4 μL lysate to PCR. When clusters are analyzed,the least reliable data (often derived from overlapping clusters) isremoved from the analysis results. Therefore, the raw data is filteredto remove any reads that do not meet the overall quality as measured bya chastity filter. The chastity of a base call is calculated as theratio of the brightest intensity divided by the sum of the brightest andsecond brightest intensities. For example, clusters “pass filter (PF)”if no more than one base call in the first 25 cycles has a chastity of<0.6. When sequencing reads are aligned to the reference genome, forexample the human genome, the first 32 bases of the read are matched toa position in the human genome and an alignment is made so long as thereare no more than 2 mismatches within the 32 base seed. Reads that couldbe aligned in more than one position in the genome are still classifiedas aligning, but they align with a low alignment score. Bases are scoredfor quality based on a combination of metrics, including their chastityscore, whether they follow a known difficult sequence and where theyfall in the sequencing read. For example, the percent of bases with a Qscore of 30 or more can be reported which means that there is aprobability of 1 in 1000 that this base call is incorrect. The coveragemetric reported indicates the number of times a particular region of thegenome has been covered by sequencing reads. The diversity metricreported is an estimated number of unique fragments present in theoriginal sequencing library. AT and GC drop out metrics refer to thedifference in the AT or GC content in the reads versus the reference.

In this experiment, an aliquot of the DNA amplification product wasloaded onto a flow cell with capture probes for clonal amplification(cluster generation) and sequencing (MiSeq). Each point on plot 200represents an amplicon and shows the percent GC content of the ampliconas a function of clusters per amplicon. In the targeted amplificationassay, for example, a uniformity value can be quoted. This metricreports the percentage of amplicons that are covered within 0.2× meancoverage, i.e., it would not include amplicons sequenced at a frequencyof less than 20% of the mean coverage of all the amplicons. Thesequencing metrics are shown in Table 1. The data show that allamplicons are covered. In this sequencing example, the cluster densityis 1626,000 clusters per mm² of flow cell surface, 80.54% of theclusters pass filters, 99.4% of the passing filters clusters align tothe human genome and 94.7% of the passing filters clusters have aquality of greater or equal to Q30.

TABLE 1 MiSeq metrics Density 1626 K/mm² PF 80.54% Align 99.4% >=Q3094.7%

FIGS. 3A and 3B show the results from a simplified method of preparing awhole blood sample for targeted DNA amplification, where an aliquot (10μL) of a whole blood sample is mixed with water (90 μL) prior toamplification (2 μL lysate used for PCR amplification process, sameprocess as for FIG. 2). For example, targeted DNA amplification may beperformed according to the methods described in the WO2010/038042publication, the WO2011/025477 publication, PCT applicationPCT/US2014/071263, filed Dec. 18, 2014, and/or PCT applicationPCT/EP2014/079145, filed Dec. 23, 2014, each of which is incorporatedherein by reference in its entirety. Here, the incubation time was onlyapproximately 2 minutes, which effectively was the time taken to mix theblood sample and water together and then prepare the amplification step.

FIGS. 3A and 3B show a plot 300 of amplicon size and a plot 350 ofpercent GC content, respectively, from a targeted DNA amplificationassay performed on a whole blood sample prepared by dilution with water.In this example, 10 μL of a whole blood sample was mixed with 90 μL ofwater. In this sample preparation protocol, water is acting as a lysingagent. A 2 μL aliquot of the sample (lysate) was amplified by targetedDNA amplification. An aliquot of the DNA amplification product wasloaded onto a flow cell with capture probes for clonal amplification(cluster generation) and sequencing (MiSeq). Each point on plots 300 and350 represent an amplicon.

These results show that the DNA preparation with water alone as thelysis reagent gives comparable results as the DNA preparation using anon-water lysis reagent (e.g. lysis reagent from “Extract-N-Amp” BloodPCR kit, Sigma).

FIG. 4 shows a data table 400 of the sequencing metrics for the targetedDNA amplification assay of FIGS. 3A and 3B. The data of FIGS. 3A, 3B,and 4 show that dilution of whole blood sample in water is sufficientfor preparing a blood sample for targeted DNA amplification andsubsequent cluster generation and sequencing.

In yet another embodiment of the invention, a whole blood sample is useddirectly for targeted DNA amplification. For example, targeted DNAamplification may be performed according to the methods described in theWO2010/038042 publication, the WO2011/025477 publication, the U.S. PCTapplication PCT/US2014/071263, filed Dec. 18, 2014, and/or PCTapplication PCT/EP2014/079145, filed Dec. 23, 2014, each of which isincorporated herein by reference in its entirety.

In this embodiment, an enzyme is used to lyse the cells (e.g. bloodcells) directly, as part of the amplification process.

A particularly preferred enzyme is Phusion DNA polymerase (New EnglandBiolabs, Thermo Scientific®), a High-Fidelity DNA Polymerase.High-Fidelity DNA Polymerases are important for applications in whichthe DNA sequence needs to be correct after amplification. PhusionHigh-Fidelity DNA Polymerase offers both high fidelity and robustperformance, and thus can be used for all PCR applications. Itsstructure, a novel Pyrococcus-like enzyme fused with aprocessivity-enhancing domain, increases fidelity and speed. Phusion DNAPolymerase is using for cloning and can be used for long or difficultamplicons. With an error rate supposedly of >50-fold lower than that ofTaq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNAPolymerase, Phusion is allegedly one of the most accurate thermostablepolymerases available. Phusion DNA Polymerase possesses 5′→3′ polymeraseactivity, 3′→5′ exonuclease activity and will generate blunt-endedproducts.

FIG. 5 shows panels of clusters generated from amplicons generateddirectly from a whole blood sample by targeted DNA amplification. Inthis example, 2 μL of whole blood is mixed directly with 48 μL of PCRmix containing Phusion enzyme (50 μL reaction volume). Clustergeneration was performed using 10, 1, 0.1, 0.01, and 0.001 μL ofamplified PCR products. PhiX (5 pM), and target sequences positivecontrols were used as positive controls.

FIG. 6 illustrates a flow diagram of another aspect of the presentinvention, in this case a method 600 of preparing a sample (e.g. wholeblood) for construction of a tagmented DNA library (e.g. via Nextera™,Illumina, Inc.). Method 600 includes, but is not limited to, thefollowing steps.

At a step 610, a sample (e.g. whole blood) is obtained or provided.

At a step 615, an aliquot (e.g. 2 μL) of whole blood is mixed with water(e.g. 12 μL).

At a step 620, as a specific step when the amplification processinvolves tagmentation, proteinase K is added to the blood sample todisrupt the chromatin. If proteinase K is not added, then the DNA doesnot fully unfold (i.e. is remains associated with histones) and largersequences of DNA ultimately become tagmented. In one example, 1 μL ofproteinase K is added to the 14 μL blood+water sample and incubated atapproximately 56° C. for 20 minutes. The proteinase K is subsequentlyinactivated by heating the sample at 70° C. for 10 minutes.

This reaction is advantageously performed as a single pot reaction,again not requiring any purification of the DNA prior to thetagmentation steps.

At a step 625, the sample is tagmented using a modified Nextera reactionto generate a tagmented DNA library. In one example, a tagmentationprotocol is based on a fast lysis protocol, Nextera kit #1502811, and anindexed kit #15028216. Briefly, 25 μL tagment DNA buffer (TD) and 10 μLtagment DNA enzyme (TDE1) are added to a fast lysis sample and incubatedfor 5 minutes at 55° C. The sample is then cooled on ice. The sample ispurified using a Zymo purification column and eluted to 25 μL. A 20 μLaliquot of the purified sample is PCR amplified using 5 μL of both indexprimers (e.g., indexes N702 and N507), 15 μL Nextera PCR mastermix(NPM), and 5 μL PCR primer cocktail (PPC). Thermal amplification isperformed according to manufacturer's recommendation. The sample volumeis adjusted (if necessary) to 30 μL with resuspension buffer (RSB) andpurified using SPRI beads.

The purified library is eluted from the SPRI beads with 32.5 μL RSB. Thefragment size distribution in the library and DNA concentration aredetermined.

FIGS. 7A and 7B show a bar graph 700 of sequencing depth per chromosomeand a plot 750 of the size distribution, respectively, of a tagmentedDNA library prepared according to method 600 of FIG. 6. An aliquot ofthe tagmented DNA library was loaded onto a flow cell for clonalamplification (cluster generation) and sequencing (MiSeq). Thesequencing metrics are shown in Table 2. In this sequencing example, thecluster density is 424,000 clusters per mm² of flow cell surface, 96.65%of the clusters pass filters, 93.45% of the passing filters clustersalign to the human genome, and 98.2% of the passing filters clustershave a quality of greater or equal to Q30. The library diversity is 4.63billion and the depth of coverage of the human genome is 0.15×. GC andAT drop out is 0.35 and 16.31 respectively.

TABLE 2 MiSeq metrics Density 424 K/mm² PF 96.65% Align 93.45% >=Q3098.2% Div 4.63 billion Depth 0.15x GD d.o. 0.35 AT d.o. 16.31

As used herein, the term “tagmentation” refers to the modification ofDNA by a transposome complex comprising transposase enzyme complexedwith adaptors comprising transposon end sequence. Tagmentation resultsin the simultaneous fragmentation of the DNA and ligation of theadaptors to the 5′ ends of both strands of duplex fragments. Following apurification step to remove the transposase enzyme, additional sequencescan be added to the ends of the adapted fragments, for example by PCR,ligation, or any other suitable methodology known to those of skill inthe art.

The method of the invention can use any transposase that can accept atransposase end sequence and fragment a target nucleic acid, attaching atransferred end, but not a non-transferred end. A “transposome” iscomprised of at least a transposase enzyme and a transposase recognitionsite. In some such systems, termed “transposomes”, the transposase canform a functional complex with a transposon recognition site that iscapable of catalyzing a transposition reaction. The transposase orintegrase may bind to the transposase recognition site and insert thetransposase recognition site into a target nucleic acid in a processsometimes termed “tagmentation”. In some such insertion events, onestrand of the transposase recognition site may be transferred into thetarget nucleic acid.

In standard sample preparation methods, each template contains anadaptor at either end of the insert and often a number of steps arerequired to both modify the DNA or RNA and to purify the desiredproducts of the modification reactions. These steps are performed insolution prior to the addition of the adapted fragments to a flowcellwhere they are coupled to the surface by a primer extension reactionthat copies the hybridized fragment onto the end of a primer covalentlyattached to the surface. These ‘seeding’ templates then give rise tomonoclonal clusters of copied templates through several cycles ofamplification.

The number of steps required to transform DNA into adaptor-modifiedtemplates in solution ready for cluster formation and sequencing can beminimized by the use of transposase mediated fragmentation and tagging.

In some embodiments, transposon based technology can be utilized forfragmenting DNA, for example as exemplified in the workflow for Nextera™DNA sample preparation kits (Illumina, Inc.) wherein genomic DNA can befragmented by an engineered transposome that simultaneously fragmentsand tags input DNA (“tagmentation”) thereby creating a population offragmented nucleic acid molecules which comprise unique adaptersequences at the ends of the fragments.

Some embodiments can include the use of a hyperactive Tn5 transposaseand a Tn5-type transposase recognition site (Goryshin and Reznikoff, J.Biol. Chem., 273:7367 (1998)), or MuA transposase and a Mu transposaserecognition site comprising R1 and R2 end sequences (Mizuuchi, K., Cell,35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Anexemplary transposase recognition site that forms a complex with ahyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase, EpicentreBiotechnologies, Madison, Wis.).

More examples of transposition systems that can be used with certainembodiments provided herein include Staphylococcus aureus Tn552 (Colegioet al., J. Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol.Microbiol., 43: 173-86, 2002), Ty 1 (Devine & Boeke, Nucleic Acids Res.,22: 3765-72, 1994 and International Publication WO 95/23875), TransposonTn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: CurrTop Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner N, etal., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner transposase(Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr.Topics Microbiol. Immunol., 204: 125-43, 1996), P Element (Gloor, G B,Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol.Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo &Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses(Brown, et al., Proc Natl Acad Sci USA, 86:2525-9, 1989), andretrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34,1989). More examples include ISS, Tn10, Tn903, IS911, and engineeredversions of transposase family enzymes (Zhang et al., (2009) PLoS Genet.5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol.Methods 71:332-5).

Sequencing Directly from FFPE

Also presented herein are methods for preparing a sequencing librarydirectly from an FFPE sample. In some embodiments, the method comprisesamplification of nucleic acid from the FFPE sample without performing axylene deparafinization step. In some embodiments, the method comprisesamplification of nucleic acid from the FFPE sample without performing aseparate extraction step. In some embodiments, amplification isperformed directly in a vessel comprising the FFPE sample.

FIG. 8 illustrates a flow diagram of an example of a method 800 ofpreparing a tissue sample (e.g. FFPE sample) for targeted DNAamplification and subsequent sequencing. Targeted DNA amplification may,for example, be performed according to the methods described in theWO2010/038042 publication, the WO2011/025477 publication, PCTapplication PCT/US2014/071263, filed Dec. 18, 2014, and/or PCTapplication PCT/EP2014/079145, filed Dec. 23, 2014, each of which isincorporated herein by reference in its entirety. Method 800 includes,but is not limited to, the following steps.

At a step 810, a tissue sample (e.g. slice of an FFPE sample) isobtained. In one example the FFPE sample is a paraffin-embedded cellculture sample. In another example, the FFPE sample is a tumor sample ora normal tissue sample.

At a step 815, DNA from the sample is extracted. In one example, theQuickExtract buffer (available from EpiCentre) is used to extract DNAfrom the FFPE sample. In this example, 100 μL of QuickExtract buffer isadded to the FFPE sample in a microfuge tube, the tube is vortexed, andincubated for 1 hour at 56° C., followed by a 2 minute incubation at 98°C.

At a step 820, the DNA in the sample is quantitated. There are severalmethods known to the skilled person to quantitate the averageconcentration of DNA present in the mixture, includingspectrophotometric quantification and UV fluorescence in the presence ofa dye.

At a step 825, targeted DNA amplification is performed on the extractedDNA sample. In one example, targeted DNA amplification is performedusing 10 ng of extracted DNA.

Preferably, by ‘extracted DNA’ it is meant the DNA that has becomeaccessible through lysis of the cellular sample. Thus, 10 ng ofextracted DNA may actually require a larger volume of lysate to beprovided.

At a step 830, the amplified DNA is diluted (e.g., 1/20) and loaded ontoa flow cell prepared with capture probes for cluster generation andsequencing (MiSeq).

In one example, method 800 of FIG. 8 is used to prepare a colon tumorFFPE sample for targeted amplification.

FIG. 9A shows a bar graph 900 of the Q score metric and a data table 920of sequencing metrics for amplicons generated from DNA prepared from acolon tumor FFPE sample according to method 800 of FIG. 8. FIG. 9B showsa bar graph 940 of the Q score metric and a data table 960 of sequencingmetrics for amplicons generated from DNA prepared from a colon tumorFFPE sample using a traditional QiaAmp DNA purification kit.

FIGS. 10A and 10B show a data table 1000 of sequencing metrics and aplot 1050 of amplicon clusters for the QiaAmp prepared DNA and theQuickExtract prepared DNA of FIGS. 9A and 9B. In this sequencing exampleof the quick extract method, the cluster density is 305,000 clusters permm² of flow cell surface, 97% of the clusters pass filters, 97.9% of thepassing filters clusters align to the human genome, and 98.7% of thepassing filters clusters have a quality of greater or equal to Q30.

As can be seen in FIGS. 9 and 10, in accordance with the methods of thepresent invention which do not employ DNA purification prior toamplification the results are closely comparable to the more classicmethod of purifying DNA prior to amplification. Thus, it appears thatthe methods of the present invention are beneficial in providingcomparable results in quicker time and a lower cost than traditionalpurification methods.

In another example, the extraction of DNA from an FFPE sample andtargeted amplification may be combined in a single reaction tube. Forexample, following the method described above in relation to FIG. 8, theproteinase K in the QuickExtract buffer is first heat inactivated andthen combined with targeted DNA amplification mix in a 40:60 ratio. AHorizon FFPE slice is added to this buffer and incubated for 1 hour at56° C. followed by 2 minutes at 98° C. The sample is then amplified bythermocycling. The data is shown in Table 3. In this sequencing example,the cluster density is 941,000 clusters per mm² of flow cell surface,93.6% of the clusters pass filters, 99.5% of the passing filtersclusters align to the human genome, and 97.8% of the passing filtersclusters have a quality of greater or equal to Q30.

TABLE 3 MiSeq metrics Density 941 K/mm² PF 93.6% Align 99.5% >=Q30 97.8%uniformity 96.3%

In another example, a Horizon FFPE slice is added to the targeted DNAamplification PCR mix followed directly by thermocycling (optionally noincubation). The data is shown in Table 4. In this sequencing example,the cluster density is 772,000 clusters per mm² of flow cell surface,94.4% of the clusters pass filters, 99.4% of the passing filtersclusters align to the human genome, and 98% of the passing filtersclusters have a quality of greater or equal to Q30.

TABLE 4 MiSeq metrics Density 772 K/mm² PF 94.4% Align 99.4% >=Q30  98%uniformity 91.3%

FIGS. 11A and 11B show a plot 1100 of amplicon size and a plot 1150 ofpercent GC content, respectively, from the direct targeted amplificationof an FFPE slice (no incubation). Each point on plots 1100 and 1150represent an amplicon.

These results are again comparable to more traditional methods involvingDNA purification.

Optionally, the present invention can also include the step ofsequencing the DNA sequence after amplification. This is preferablyconducted via high throughput sequencing methods.

In some embodiments, as illustrated in FIG. 25, a sequencing library canbe prepared directly from an FFPE sample. FIG. 25 shows samplepreparation workflows for preparing a DNA library from an FFPE sampleusing modifications of the workflow set forth in FIG. 8. Typical methodsusing QlAamp tools rely on a xylene deparafinization step, followed byincubation with proteinase K and heating to remove crosslinking. InFIGS. 26-27, such embodiments are referred to as “Qiagen purification”.Typical methods using xylene deparafinization and proteinase K lysis canrequire close to 3 hours of processing, as indicated in FIGS. 25-27 (“2h 45 m”).

The methods presented herein provide sequencing-ready libraries withminimal handling, thus reducing processing time and removingopportunities for user error and sample loss.

In some embodiments, a sequencing library is prepared by incubating anFFPE sample with an extraction buffer, such as, for example QuickExtractbuffer (Epicentre) or another suitable extraction buffer. One suitablebuffer is set forth in Table 5 below.

TABLE 5 Tris HCL pH 7.5 50 mM EDTA 1 mM Proteinase K 100 mg/ml 0.5 mg/ml10% Triton X100 0.5%

In some embodiments, as indicated in FIG. 25, an FFPE sample isincubated in an extraction buffer, such as a buffer comprising thecomponents in Table 5. In some embodiments, following an extractionstep, total DNA is quantified, and a portion of the extracted DNA isused as input for a targeted amplification reaction, such as PCRamplification as described hereinabove. In FIGS. 26-27, such embodimentsare referred to as “1 h 20 min workflow”.

Thus, in embodiments presented herein, DNA obtained from FFPE issubjected to targeted amplification, and the amplicons generated aresequenced by, for example, SBS methodology. In some such embodiments,because DNA obtained from FFPE is not purified prior to placing on asequencing instrument, the sequencing apparatus (flowcells and the like)will comprise components from the FFPE sample besides DNA. Examples ofFFPE components include, for example, formalin, paraffin, cellularcomponents, protein, extracellular matrix components, collagen, tissuedebris, and the like.

Thus, presented herein is a method of performing a clustering reactionon a solid surface, wherein the clustering reaction is performed in thepresence of one or more of formalin, paraffin, cellular components,protein, extracellular matrix components, collagen, and tissue debris.In some embodiments, the clustering reaction is performed in thepresence of at least 0.001 μg paraffin. In some embodiments, theclustering reaction is performed in the presence of at least 0.01 pg,0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 rig, 10 μg, 100 rig,or at least 1 mg paraffin.

Presented herein is a flowcell comprising immobilized amplificationprimers and one or more of formalin, paraffin, cellular components,protein, extracellular matrix components, collagen, and tissue debris.In some embodiments, the flowcell comprises at least 0.001 pg paraffin.In some embodiments, the flowcell comprises at least 0.01 pg, 0.1 pg, 1pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 rig, 100 rig, or atleast 1 mg paraffin.

Thus, presented herein is a method of performing a clustering reactionon a solid surface, wherein the clustering reaction is performed in thepresence of proteinase K. In some embodiments, the clustering reactionis performed in the presence of at least 0.001 pg proteinase K. In someembodiments, the clustering reaction is performed in the presence of atleast 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg,10 μg, 100 μg, or at least 1 mg proteinase K.

Presented herein is a flowcell comprising immobilized amplificationprimers and proteinase K. In some embodiments, the flowcell comprises atleast 0.001 pg proteinase K. In some embodiments, the flowcell comprisesat least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1μg, 10 μg, 100 μg, or at least 1 mg proteinase K.

In some embodiments, as indicated in FIG. 25, PCR amplification isperformed without performing DNA quantification. In FIGS. 26-27, suchembodiments are referred to as “1 h 2 min workflow”.

In all embodiments depicted in FIG. 25, an aliquot of the amplificationreaction was placed directly into a MiSeq flowcell and clustering,followed by SBS sequencing, was performed according to manufacturerinstructions.

In typical embodiments, an FFPE slice is about 10 μm in thickness. Insome embodiments, an FFPE slice can be as thin as around 1, 2, 3, 4, 5,6, 7, 8, 9 or more than 9 μm in thickness. Thus, in a 10 μm thick FFPEslice, paraffin ranging from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 mg of paraffincan be present. Similarly, in a 10 μm thick FFPE slice, tissue rangingfrom 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more than 20 mg of tissue can be present. Thesecomponents are transferred into a sequencing flowcell in embodimentspresented herein. In some embodiments, a tissue slice is contacted with10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 μL of anextraction buffer comprising a proteinase, such as proteinase K. Ontypical extraction buffer is set forth above in Table 5.

In some embodiments, as indicated in FIG. 25, an FFPE sample placeddirectly into an amplification reaction, such as PCR amplificationbuffer, without first performing a separate extraction step. In FIGS.26-27, such embodiments are referred to as “2 min workflow”.

FIG. 26 is a data table comparing coverage uniformity obtained forsequencing libraries obtained using 4 different workflows from 3different FFPE samples. Surprisingly, clustering and sequencingperformed in the presence of one or more of proteinase K, formalin,paraffin, cellular components, protein, extracellular matrix components,collagen, and tissue debris resulted in sequencing coverage uniformitywhich is comparable to those obtained using purified DNA.

FIG. 27 shows plots of clusters that pass filter for amplicons generatedby targeted DNA amplification of FFPE samples prepared according to twomethods of FIG. 25. Surprisingly, clustering and sequencing performed inthe presence of one or more of proteinase K, formalin, paraffin,cellular components, protein, extracellular matrix components, collagen,and tissue debris resulted in sequencing coverage uniformity which iscomparable to those obtained using purified DNA.

Sequencing Directly from Dried Blood Spots

The inventors have also shown that amplicon sequencing can be carriedout directly from dried blood spots. In this experiment, shown in FIG.12, dried blood spots where provided on Whatman 903 filter paper with˜50-70 μL blood per spot. This is equivalent to the dried blood spotsfound on Guthrie cards used for newborn heel prick tests. In thisexperiment blood was dried down onto the paper for 1-6 days.

A 3 mm² portion was cut out from the blood spot and placed into a tube.The portion is rinsed with water which washes away protein inhibitorscontained in the blood. DNA remains bound to filter paper. In thisexample, 2×5 min 100 μL washes were carried out. Rinsed filter paperplaced into tube with PCR mix. Potentially this could be done as aone-pot reaction. PCR, clustering and sequencing is then carried out.Results can be seen in FIGS. 13A and 13B.

Experiments were also carried out to show that tagmentation based wholegenome sequencing can be carried out on dried blood spots. FIG. 14 showsthe experiment carried out both with a water wash step (top) and without(bottom). Again 3 mm² portions were cut out from the blood spot andplaced into a tube. In the top panel the portion is washed twice withwater and incubated for 5 minutes. In the bottom panel no wash stepoccurs. In both cases tagmentation, PCR (with and without filter) andsequencing then occurs with the results being shown in FIG. 15.

Bead Based Nextera (BBN)

Methods for performing bead based tagmentation, also referred to hereinas bead based Nextera™ (BBN) are described in the materials of U.S.Application Ser. No. 62/167,463, filed May 28, 2015, the content ofwhich is incorporated by reference in its entirety. Briefly, asperformed in some embodiments presented herein, BBN can be performed asfollows. 20 μL of blood sample is mixed with 20 μL of magnetic beads(100 nM TSM) and 10 μL of tagment buffer (TD) and incubated for 15minutes at 55° C. 12.5 μL of NT buffer is added to the sample andincubated for 5 minutes at room temperature. The sample is placed on amagnet, supernatant removed. The beads were washed three times with 100μL of HT2 buffer for each wash step. The sample is PCR amplified using 5μL of both index primers (e.g., indexes N702 and N507), 15 μL NexteraPCR mastermix (NPM), and 5 μL PCR primer cocktail (PPC). Thermalamplification is performed according to manufacturer's recommendation((e.g. 5 PCR cycles). The tube containing the beads and sample is placedon magnet and supernatant purified using ZYMO columns (Zymo Research)and SPRI beads.

A comparison of Bead Based Nextera with other whole blood librarypreparation methods was performed. FIGS. 16, 17 and 18 show sequencingmetrics and sequencing depth per chromosome of a tagmented DNA libraryprepared according to three different methods. For the data shown inFIG. 16, a library from whole blood was prepared generally according tomethod 600 of FIG. 6. In particular, 2 μL whole blood was mixed with 12μL water and then incubated with proteinase K at 56° C. for 10 minutes.Tagmentation reagents were then added to the lysate and tagmentationperformed as described above. An aliquot of the tagmented DNA librarywas loaded onto a flow cell for clonal amplification (clustergeneration) and sequencing (MiSeq). For the data shown in FIG. 17, amodification of the above protocol was made. Specifically, cellulardebris was pelleted after lysis with water, and prior to incubation withproteinase K. After proteinase K incubation, the sample was centrifugedto remove solid debris, and then bead based Nextera was performed asdescribed above. For the data shown in FIG. 18, a 3 mm² piece of filterpaper having a dried blood spot was rinsed with water for 15 minutes,and the solubilized DNA was removed and tagmentation performed asdescribed above.

The sequencing metrics are shown in Tables for the 3 methodsrespectively in FIGS. 16, 17 and 18. In this sequencing example, thecluster densities are 4.11 billion, 2.25 billion and 0.85 billionclusters per mm² of flow cell surface for the three workflowsrespectively. GC and AT drop out are also given in the tables for thethree methods, respectively.

FIG. 19 shows a comparison of the sequencing metrics for three BBNsamples (BBN1, 2, 3) as compared to sequencing metrics of purified gDNAcontrols and dried blood spots, prepared as described above. In thissequencing example, the cluster density, % of the clusters passingfilters, % of the passing filters clusters align to the human genome,and % of the passing filters clusters having a quality of greater orequal to Q30 are comparable for BBN samples, dried blood, and purifiedgDNA samples. FIGS. 20 and 21 show additional comparisons of coverage ofgene panels of interest (FIG. 20) and genotype precision and recallrelated to SNP concordance (FIG. 21), indicating that data output forthe three methods are comparable.

Non-Tagmented Libraries

In some embodiments presented herein, a method for library preparationfrom whole blood can involve adapter ligation, thus avoiding the needfor tagmentation reagents. As shown in FIG. 22, a standard librarypreparation method, designated as “TruSeq Nano” includes DNA extraction,shearing (Covaris), cleanup and size selection (SPRI), followed by endrepair, A-tailing and adapter ligation. It has been surprisinglydiscovered that modifications to this workflow can greatly improvelibrary preparation from whole blood samples, as demonstrated by severalmetrics.

In one embodiment, library preparation directly from blood usingmodifications to the Illumina TruSeq Nano kit is described. Briefly, asillustrated by WF-2 in FIG. 22, 16 μl of blood was mixed with 36 μLwater and 8 μL of Proteinase K and incubated at 56° C. for 10 minutesfollowed by 70° C. for 10 minutes. Samples were spun down at 10,000 gfor 1 minute followed by shearing using Covaris sonication. The samplewas then purified using SPRI beads and followed by end repair,A-tailing, ligation and PCR amplification according to manufacturerrecommendations for TruSeq Nano (Illumina, Inc). In another embodiment,illustrated in FIG. 22 as workflow (WF-4), 16 μL of blood is mixed with104 μL RSB (10 mM Tris pH 7.0) followed by shearing on COVARIS. 8 μL ofProteinase K was added to the sheared blood sample and incubated at 56°C. for 10 minutes followed by 70° C. for 10 minutes. Samples were spundown at 10,000 g for 1 minute. Sample was purified using SPRI beads andfollowed by end repair, A-tailing, ligation and PCR amplificationaccording to manufacturer recommendations for TruSeq Nano (Illumina,Inc).

FIG. 23 shows the GC bias profiles of libraries prepared from the threeTrueSeq Nano workflows described above for whole blood. As shown in FIG.23, WF2 exhibited a better GC bias profile, representing an unexpectedfinding and a significant improvement over the control DNA samplepreparation.

FIG. 24 shows that the diversity of WF-2 and WF-4 are higher than thatof control sample.

In summary, Workflows 2 and 4 (WF-2, WF-4) resulted in overall bettercoverage and callability. Both workflows avoid the need for DNAextraction, offering significant savings in cost and time.

Sequencing Methods

In principle, next generation sequencing (NGS) is similar toSanger-based, or CE sequencing. The bases of a small fragment of DNA aresequentially identified from signals emitted as each fragment isre-synthesized from a DNA template strand. NGS extends this processacross millions of reactions in a massively parallel fashion, ratherthan being limited to a single or a few DNA fragments. This advanceenables rapid sequencing of large stretches of DNA, with the latestinstruments capable of producing hundreds of gigabases of data in asingle sequencing run. To illustrate how this process works, consider asingle genomic DNA (gDNA) sample. The gDNA is first fragmented into alibrary of small segments and sequenced. The newly identified strings ofbases, called reads, are then reassembled using a known reference genomeas a scaffold (resequencing), or assembled together using advancedcomputational techniques if no reference genome is available (de novosequencing). The full set of aligned reads reveals the entire genomicsequence of the sample. Once the sample library is prepared, all of thesequencing steps through data analysis can be performed on a singleinstrument, facilitating rapid turnaround with minimal hands-on time.

With NGS, researchers can start directly from a gDNA or cDNA library.The DNA fragments are then ligated to specific oligonucleotide adaptersneeded to perform the sequencing biochemistry, requiring as little as 90minutes with Illumina's Nextera® technology. In contrast, CE-basedSanger sequencing requires genomic DNA to be fragmented first and clonedinto either bacterial artificial chromosomes (BACs) or yeast artificialchromosomes (YACs). Then, each BAC/YAC must be further subcloned into asequencing vector and transformed into the appropriate microbial host.Template DNA is then purified from individual colonies or plaques priorto sequencing. This process can take days or even weeks to complete.

Sequencing by Synthesis (SBS) Technology

Illumina's sequencing instruments and reagents support massivelyparallel sequencing using a proprietary method that detects single basesas they are incorporated into growing DNA strands.

SBS Chemistry

A fluorescently labeled reversible terminator is imaged as each dNTP isadded, and then cleaved to allow incorporation of the next base. Sinceall four reversible terminator-bound dNTPs are present during eachsequencing cycle, natural competition minimizes incorporation bias. Theend result is true base-by-base sequencing that enables the industry'smost accurate data for a broad range of applications.

The methods described herein can be used in conjunction with a varietyof nucleic acid sequencing techniques. Particularly applicabletechniques are those wherein nucleic acids are attached at fixedlocations in an array such that their relative positions do not changeand wherein the array is repeatedly imaged. Embodiments in which imagesare obtained in different color channels, for example, coinciding withdifferent labels used to distinguish one nucleotide base type fromanother are particularly applicable. In some embodiments, the process todetermine the nucleotide sequence of a target nucleic acid can be anautomated process. Preferred embodiments include sequencing-by-synthesis(“SBS”) techniques.

SBS techniques generally involve the enzymatic extension of a nascentnucleic acid strand through the iterative addition of nucleotidesagainst a template strand. In traditional methods of SBS, a singlenucleotide monomer may be provided to a target nucleotide in thepresence of a polymerase in each delivery. However, in the methodsdescribed herein, more than one type of nucleotide monomer can beprovided to a target nucleic acid in the presence of a polymerase in adelivery.

SBS can utilize nucleotide monomers that have a terminator moiety orthose that lack any terminator moieties. Methods utilizing nucleotidemonomers lacking terminators include, for example, pyrosequencing andsequencing using γ-phosphate-labeled nucleotides, as set forth infurther detail below. In methods using nucleotide monomers lackingterminators, the number of nucleotides added in each cycle is generallyvariable and dependent upon the template sequence and the mode ofnucleotide delivery. For SBS techniques that utilize nucleotide monomershaving a terminator moiety, the terminator can be effectivelyirreversible under the sequencing conditions used as is the case fortraditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methodsdeveloped by Solexa (now Illumina, Inc.).

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of thenucleotide, such as release of pyrophosphate; or the like. Inembodiments, where two or more different nucleotides are present in asequencing reagent, the different nucleotides can be distinguishablefrom each other, or alternatively, the two or more different labels canbe the indistinguishable under the detection techniques being used. Forexample, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriateoptics as exemplified by the sequencing methods developed by Solexa (nowIllumina, Inc.).

Preferred embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, the disclosures of which are incorporatedherein by reference in their entireties). In pyrosequencing, releasedPPi can be detected by being immediately converted to adenosinetriphosphate (ATP) by ATP sulfurylase, and the level of ATP generated isdetected via luciferase-produced photons. The nucleic acids to besequenced can be attached to features in an array and the array can beimaged to capture the chemiluminscent signals that are produced due toincorporation of a nucleotides at the features of the array. An imagecan be obtained after the array is treated with a particular nucleotidetype (e.g. A, T, C or G). Images obtained after addition of eachnucleotide type will differ with regard to which features in the arrayare detected. These differences in the image reflect the differentsequence content of the features on the array. However, the relativelocations of each feature will remain unchanged in the images. Theimages can be stored, processed and analyzed using the methods set forthherein. For example, images obtained after treatment of the array witheach different nucleotide type can be handled in the same way asexemplified herein for images obtained from different detection channelsfor reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures ofwhich are incorporated herein by reference. This approach is beingcommercialized by Solexa (now Illumina Inc.), and is also described inWO 91/06678 and WO 07/123,744, each of which is incorporated herein byreference. The availability of fluorescently-labeled terminators inwhich both the termination can be reversed and the fluorescent labelcleaved facilitates efficient cyclic reversible termination (CRT)sequencing. Polymerases can also be co-engineered to efficientlyincorporate and extend from these modified nucleotides.

Preferably in reversible terminator-based sequencing embodiments, thelabels do not substantially inhibit extension under SBS reactionconditions. However, the detection labels can be removable, for example,by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. Inparticular embodiments, each cycle involves simultaneous delivery offour different nucleotide types to the array and each nucleotide typehas a spectrally distinct label. Four images can then be obtained, eachusing a detection channel that is selective for one of the fourdifferent labels. Alternatively, different nucleotide types can be addedsequentially and an image of the array can be obtained between eachaddition step. In such embodiments each image will show nucleic acidfeatures that have incorporated nucleotides of a particular type.Different features will be present or absent in the different images duethe different sequence content of each feature. However, the relativeposition of the features will remain unchanged in the images. Imagesobtained from such reversible terminator-SBS methods can be stored,processed and analyzed as set forth herein. Following the image capturestep, labels can be removed and reversible terminator moieties can beremoved for subsequent cycles of nucleotide addition and detection.Removal of the labels after they have been detected in a particularcycle and prior to a subsequent cycle can provide the advantage ofreducing background signal and crosstalk between cycles. Examples ofuseful labels and removal methods are set forth below.

In particular embodiments some or all of the nucleotide monomers caninclude reversible terminators. In such embodiments, reversibleterminators/cleavable fluors can include fluor linked to the ribosemoiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005),which is incorporated herein by reference). Other approaches haveseparated the terminator chemistry from the cleavage of the fluorescencelabel (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), whichis incorporated herein by reference in its entirety). Ruparel et aldescribed the development of reversible terminators that used a small 3′allyl group to block extension, but could easily be deblocked by a shorttreatment with a palladium catalyst. The fluorophore was attached to thebase via a photocleavable linker that could easily be cleaved by a 30second exposure to long wavelength UV light. Thus, either disulfidereduction or photocleavage can be used as a cleavable linker. Anotherapproach to reversible termination is the use of natural terminationthat ensues after placement of a bulky dye on a dNTP. The presence of acharged bulky dye on the dNTP can act as an effective terminator throughsteric and/or electrostatic hindrance. The presence of one incorporationevent prevents further incorporations unless the dye is removed.Cleavage of the dye removes the fluor and effectively reverses thetermination. Examples of modified nucleotides are also described in U.S.Pat. Nos. 7,427,673, and 7,057,026, the disclosures of which areincorporated herein by reference in their entireties.

Additional exemplary SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentApplication Publication No. 2007/0166705, U.S. Patent ApplicationPublication No. 2006/0188901, U.S. Pat. No. 7,057,026, U.S. PatentApplication Publication No. 2006/0240439, U.S. Patent ApplicationPublication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S.Patent Application Publication No. 2005/0100900, PCT Publication No. WO06/064199, PCT Publication No. WO 07/010,251, U.S. Patent ApplicationPublication No. 2012/0270305 and U.S. Patent Application Publication No.2013/0260372, the disclosures of which are incorporated herein byreference in their entireties.

Some embodiments can utilize detection of four different nucleotidesusing fewer than four different labels. For example, SBS can beperformed utilizing methods and systems described in the incorporatedmaterials of U.S. Patent Application Publication No. 2013/0079232. As afirst example, a pair of nucleotide types can be detected at the samewavelength, but distinguished based on a difference in intensity for onemember of the pair compared to the other, or based on a change to onemember of the pair (e.g. via chemical modification, photochemicalmodification or physical modification) that causes apparent signal toappear or disappear compared to the signal detected for the other memberof the pair. As a second example, three of four different nucleotidetypes can be detected under particular conditions while a fourthnucleotide type lacks a label that is detectable under those conditions,or is minimally detected under those conditions (e.g., minimal detectiondue to background fluorescence, etc). Incorporation of the first threenucleotide types into a nucleic acid can be determined based on presenceof their respective signals and incorporation of the fourth nucleotidetype into the nucleic acid can be determined based on absence or minimaldetection of any signal. As a third example, one nucleotide type caninclude label(s) that are detected in two different channels, whereasother nucleotide types are detected in no more than one of the channels.The aforementioned three exemplary configurations are not consideredmutually exclusive and can be used in various combinations. An exemplaryembodiment that combines all three examples, is a fluorescent-based SBSmethod that uses a first nucleotide type that is detected in a firstchannel (e.g. dATP having a label that is detected in the first channelwhen excited by a first excitation wavelength), a second nucleotide typethat is detected in a second channel (e.g. dCTP having a label that isdetected in the second channel when excited by a second excitationwavelength), a third nucleotide type that is detected in both the firstand the second channel (e.g. dTTP having at least one label that isdetected in both channels when excited by the first and/or secondexcitation wavelength) and a fourth nucleotide type that lacks a labelthat is not, or minimally, detected in either channel (e.g. dGTP havingno label).

Further, as described in the incorporated materials of U.S. PatentApplication Publication No. 2013/0079232, sequencing data can beobtained using a single channel. In such so-called one-dye sequencingapproaches, the first nucleotide type is labeled but the label isremoved after the first image is generated, and the second nucleotidetype is labeled only after a first image is generated. The thirdnucleotide type retains its label in both the first and second images,and the fourth nucleotide type remains unlabeled in both images.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate oligonucleotides andidentify the incorporation of such oligonucleotides. Theoligonucleotides typically have different labels that are correlatedwith the identity of a particular nucleotide in a sequence to which theoligonucleotides hybridize. As with other SBS methods, images can beobtained following treatment of an array of nucleic acid features withthe labeled sequencing reagents. Each image will show nucleic acidfeatures that have incorporated labels of a particular type. Differentfeatures will be present or absent in the different images due thedifferent sequence content of each feature, but the relative position ofthe features will remain unchanged in the images. Images obtained fromligation-based sequencing methods can be stored, processed and analyzedas set forth herein. Exemplary SBS systems and methods which can beutilized with the methods and systems described herein are described inU.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize nanopore sequencing (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”. Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). Data obtained from nanopore sequencing can be stored,processed and analyzed as set forth herein. In particular, the data canbe treated as an image in accordance with the exemplary treatment ofoptical images and other images that is set forth herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and7,211,414 (each of which is incorporated herein by reference) ornucleotide incorporations can be detected with zero-mode waveguides asdescribed, for example, in U.S. Pat. No. 7,315,019 (which isincorporated herein by reference) and using fluorescent nucleotideanalogs and engineered polymerases as described, for example, in U.S.Pat. No. 7,405,281 and U.S. Patent Application Publication No.2008/0108082 (each of which is incorporated herein by reference). Theillumination can be restricted to a zeptoliter-scale volume around asurface-tethered polymerase such that incorporation of fluorescentlylabeled nucleotides can be observed with low background (Levene, M. J.et al. “Zero-mode waveguides for single-molecule analysis at highconcentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al.“Parallel confocal detection of single molecules in real time.” Opt.Lett. 33, 1026-1028 (2008); Korlach, J. et al. “Selective aluminumpassivation for targeted immobilization of single DNA polymerasemolecules in zero-mode waveguide nano structures.” Proc. Natl. Acad.Sci. USA 105, 1176-1181 (2008), the disclosures of which areincorporated herein by reference in their entireties). Images obtainedfrom such methods can be stored, processed and analyzed as set forthherein.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US 2009/0026082 A1; US2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, each ofwhich is incorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readilyapplied to substrates used for detecting protons. More specifically,methods set forth herein can be used to produce clonal populations ofamplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In particular embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate. This allows convenient delivery of sequencingreagents, removal of unreacted reagents and detection of incorporationevents in a multiplex manner. In embodiments using surface-bound targetnucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to asurface in a spatially distinguishable manner. The target nucleic acidscan be bound by direct covalent attachment, attachment to a bead orother particle or binding to a polymerase or other molecule that isattached to the surface. The array can include a single copy of a targetnucleic acid at each site (also referred to as a feature) or multiplecopies having the same sequence can be present at each site or feature.Multiple copies can be produced by amplification methods such as, bridgeamplification or emulsion PCR as described in further detail below.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.

An advantage of the methods set forth herein is that they provide forrapid and efficient detection of a plurality of target nucleic acid inparallel. Accordingly the present disclosure provides integrated systemscapable of preparing and detecting nucleic acids using techniques knownin the art such as those exemplified above. Thus, an integrated systemof the present disclosure can include fluidic components capable ofdelivering amplification reagents and/or sequencing reagents to one ormore immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell canbe configured and/or used in an integrated system for detection oftarget nucleic acids. Exemplary flow cells are described, for example,in US 2010/0111768 A1 and U.S. Ser. No. 13/273,666, each of which isincorporated herein by reference. As exemplified for flow cells, one ormore of the fluidic components of an integrated system can be used foran amplification method and for a detection method. Taking a nucleicacid sequencing embodiment as an example, one or more of the fluidiccomponents of an integrated system can be used for an amplificationmethod set forth herein and for the delivery of sequencing reagents in asequencing method such as those exemplified above. Alternatively, anintegrated system can include separate fluidic systems to carry outamplification methods and to carry out detection methods. Examples ofintegrated sequencing systems that are capable of creating amplifiednucleic acids and also determining the sequence of the nucleic acidsinclude, without limitation, the MiSeq™ platform (Illumina, Inc., SanDiego, Calif.) and devices described in U.S. Ser. No. 13/273,666, whichis incorporated herein by reference.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of thepresent disclosure. Other embodiments having different structures andoperations do not depart from the scope of the present disclosure. Theterm “the invention” or the like is used with reference to certainspecific examples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation.

What is claimed is:
 1. A method of preparing a sample for libraryamplification and subsequent amplification comprising the followingsteps: on a provided nucleic acid-containing cellular sample, (a) lysingcells of the sample with a lysis reagent to liberate nucleic acid fromwithin the cells of the cellular sample, thereby forming a lysate; (b)amplifying the nucleic acids from the lysate to form amplified nucleicacids; (c) exposing the amplified nucleic acids to a solid surface withimmobilized amplification primers, thereby immobilizing the amplifiednucleic acids on the solid surface; and (d) clonally amplifying theimmobilized, amplified nucleic acids on the solid surface to generateclusters; wherein there is no purification of the nucleic acid from thelysate prior to beginning the amplification step (b); and whereinamplifying the nucleic acid in step (b) comprises tagmentation, whereinthe sample is a blood sample, a whole blood sample, or a dried bloodsample.
 2. The method of claim 1, wherein the nucleic acid is DNA. 3.The method of claim 1, wherein the lysis reagent is water, purifiedwater, or distilled water.
 4. The method of claim 1, wherein the lysisreagent is a detergent, a non-denaturing lytic detergent, a base, anacid, and/or an enzyme.
 5. The method of claim 4, wherein the lysisreagent comprises a reducing agent, stabilizing agent,2-mercaptoethanol, or dithiothreitol.
 6. The method of claim 1, furthercomprising, prior to step (a), treating the nucleic acid-containingcellular sample with an enzyme that disrupts the structure of thenucleic acid.
 7. The method of claim 6, wherein the enzyme that disruptsthe structure of the nucleic acid is proteinase K.
 8. The method ofclaim 1, further comprising neutralizing the lysis reagent prior to theamplification step (b) to inactivate the lysis reagent.
 9. The method ofclaim 1, wherein the steps of lysing the sample and amplifying thenucleic acid contained in the lysate are conducted in a single potreaction.
 10. The method of claim 1, wherein the immobilized, amplifiednucleic acids are sequenced by high throughput sequencing or by asequence-by-synthesis protocol to ascertain their sequences.
 11. Themethod of claim 1, wherein clonally amplifying the immobilized,amplified nucleic acids on the solid surface to generate clusters isperformed in the presence of (a) proteinase K and/or (b) one or more offormalin, paraffin, cellular components, protein, extracellular matrixcomponents, collagen, and tissue debris.
 12. The method of claim 1,wherein the tagmentation is performed using bead-based tagmentation. 13.A method of preparing a sample for library amplification and subsequentamplification comprising the following steps: on a provided nucleicacid-containing cellular sample, (a) lysing cells of the sample with alysis reagent to liberate nucleic acid from within the cells of thecellular sample, thereby forming a lysate; (b) amplifying the nucleicacids from the lysate to form amplified nucleic acids; (c) exposing theamplified nucleic acids to a solid surface with immobilizedamplification primers, thereby immobilizing the amplified nucleic acidson the solid surface; and (d) clonally amplifying the immobilized,amplified nucleic acids on the solid surface to generate clusters;wherein there is no purification of the nucleic acid from the lysateprior to beginning the amplification step (b); and wherein amplifyingthe nucleic acid in step (b) comprises tagmentation, wherein the sampleis a formalin fixed paraffin embedded (FFPE) sample.
 14. The method ofclaim 13, wherein the nucleic acid is DNA.
 15. The method of claim 13,wherein the lysis reagent is water, purified water, or distilled water.16. The method of claim 13, wherein the lysis reagent is a detergent, anon-denaturing lytic detergent, a base, an acid, and/or an enzyme. 17.The method of claim 16, wherein the lysis reagent comprises a reducingagent, stabilizing agent, 2-mercaptoethanol, or dithiothreitol.
 18. Themethod of claim 13, further comprising, prior to step (a), treating thenucleic acid-containing cellular sample with an enzyme that disrupts thestructure of the nucleic acid.
 19. The method of claim 18, wherein theenzyme that disrupts the structure of the nucleic acid is proteinase K.20. The method of claim 13, further comprising neutralizing the lysisreagent prior to the amplification step (b) to inactivate the lysisreagent.
 21. The method of claim 13, wherein the steps of lysing thesample and amplifying the nucleic acid contained in the lysate areconducted in a single pot reaction.
 22. The method of claim 13, whereinthe immobilized, amplified nucleic acids are sequenced by highthroughput sequencing or by a sequence-by-synthesis protocol toascertain their sequences.
 23. The method of claim 13, wherein clonallyamplifying the immobilized, amplified nucleic acids on the solid surfaceto generate clusters is performed in the presence of at least 0.001 pgparaffin.
 24. The method of claim 13, wherein clonally amplifying theimmobilized, amplified nucleic acids on the solid surface to generateclusters is performed in the presence of (a) proteinase K and/or (b) oneor more of formalin, paraffin, cellular components, protein,extracellular matrix components, collagen, and tissue debris.
 25. Themethod of claim 13, wherein the tagmentation is performed usingbead-based tagmentation.